This proposal describes experiments investigating the molecular basis for control of DNA replication in model system derived from the plasmid Co1E1. The main tools for the investigation are plasmid copy number mutants. Replication control is mediated by a diffusible, plasmid-encoded, replicon-specific replication repressor which exerts negative control over plasmid DNA replication. A central aim of this proposal is the isolation and characterization of the replication repressor. The repressor has been mapped to a small region of the plasmid genome containing the origin of replication. We will precisely define the mutation in copy number mutants by nucleotide sequencing of mutant and wild type origin regions. A model relating replication repressor structure and function is proposed, along with experiments explicitly testing its major predictions. We will use an in vitro plasmid DNA replication system as a complementation assay for repressor purification. Methods are described for the isolation of cis-dominant, temperature-sensitive, and nonsense repressor mutants. Genetic and biochemical procedures are outlined for conditional repressor overproduction in vivo. Experiments are proposed to compare Cop plus and Cop negative plasmid replication intermediates, replication time in vivo, and the behavior of these plasmids in various mutant host backgrounds. The role of the host membrane in replication control is also explored.